1. Field of the Invention
The present invention relates to a vaccine against Eastern Equine Encephalitis (EEE) virus. More specifically the invention relates to the synthesis of an alphavirus, EEE clone that is useful as a vaccine for EEE. cDNAs coding for an infectious Eastern equine encephalitis virus are disclosed. Novel attenuating mutations and their use are described.
2. Brief Description of Related Art
Eastern equine encephalitis (EEE), Western equine encephalitis (WEE), and Venezuelan equine encephalitis virus (VEE) are members of the Alphavirus genus of the family Togaviridae. The genus is comprised of at least 27 different arthropod-borne RNA viruses that are found throughout much of the world. The viruses normally circulate among avian or rodent hosts through the feeding activities of a variety of mosquitoes. Many epizootics have occurred and they correlate with increased mosquito activity after periods of increased rainfall.
Based on comparisons of their genomic sequences, EEE, WEE and VEE appear to be closely related New World alphaviruses. All three viruses are known to cause encephalitis in humans and equines in epidemic proportions. However, EEE causes the most severe of the arboviral encephalitides in humans, with 50-75% mortality and severe neurological sequelae in survivors (Fields Virology, 4th Ed., Chapter 30 Alphaviruses, [2002] 917-962). The highest case fatality rates occur in children and the elderly. In equines, the mortality rate is substantially higher, ranging from 80-90% with most survivors having significant neurologic sequela (Weaver et al. [1989] Adv Virus Res. 37:277-328). EEE outbreaks have also been observed in pigs (Elvinger et al. [1996] J Vet Diagn Invest 8:481-484) as well as in game birds such as pheasants (Sussman et al. [1958] Ann NY Acad Sci 70:328-341) and emus (Tully et al. [1992] Avian Dis 36:808-812).
In EEE epidemics, Aedes and Coquillettidia mosquito species are thought to bridge the gap between infected birds and humans or equines. The recent advances of the Aedes albopictus mosquito in North America and its known competence as an EEE vector has increased the potential for more frequent and widespread epidemics and enzootics (Mitchell et al., [1992] Science 257: 526-527). Another significant public health concern involves infected migratory birds carrying the virus into new areas. Studies have shown that this occurs. Two South American strains of EEE virus were isolated from migratory birds captured in Mississippi in the early 1970's (Calisher et al. [1971] Am J Epidemiol 94:172-178). In 1996 an EEE equine epidemic occurred in Tamaulipas State, Mexico. This area is several hundred miles outside of the geographic range of the natural EEE vector and epidemiologists believe it was caused by infected migratory birds (Brault et al. [1999] Am J Trop Med Hyg. 61:579-586).
EEE was first recognized as an equine disease in the northeastern U.S. The virus responsible for EEE was originally isolated from the brains of infected horses involved in a 1933 outbreak in Virginia and New Jersey (Ten Broeck, C. et al. [1933] Proc. Soc. Exp. Biol. Med. 31:217-220.). EEE virus has caused localized outbreaks in equines, pigs, pheasants, emus and humans during the summer months in North America. The virus is known to be focally endemic along much of the Atlantic and Gulf Coasts of North America. It has also be found in southern Canada, the Caribbean, Central America, the eastern part of Mexico and in large sections of South America. (FIG. 2). Inland foci exist in the Great Lakes region and South Dakota in the U.S. as well as the Amazon Basin.
The current EEE vaccine for human use and veterinary applications in the U.S. is a formalin-inactivated whole virus preparation derived from the PE-6 strain (Bartelloni, et al. [1970] Am J. Trop Med Hyg. 19:123-126; Marie, et al. [1970] Am J Trop Med Hyg. 19:119-122). This preparation is currently licensed for veterinary use. For humans it is an investigative new drug (IND) and is intended for persons at risk for infection (e.g., laboratory and field investigative personnel). This inactivated vaccine is poorly immunogenic, requires multiple inoculations with frequent boosters and generally results in immunity of short duration (Pittman et al. in Vaccines, 4th Ed. (Eds. Plotkin, S. A. and Orenstein, W. A.) [2004] pgs. 991-992). Another important shortcoming is that this inactive EEE vaccine provides inadequate protection against antigenically distinct South American EEE strains [Strizki et al. [1995] Am J Trop Med Hyg. 53:564-570; Dietz et al. [1980] Am J Trop Med Hyg. 29:133-140]. Last, there are dire consequences if the PE-6 vaccine preparation is not fully inactivated. Recently, an improperly inactivated PE6 preparation is suspected of causing fatal EEE encephalitis in a California equine (Franklin et al. [2002] Emerging Infectious Diseases 8:283-288).
The scientific community considers EEE to be a virus that could potentially be used as a biological weapon. The Centers for Disease Control (CDC) has categorized EEE as a class B select agent. Scientists within the ex-Soviet Union have conducted “vaccine” research on the virus (Volchkov et al. [1991] Molekulyarnaya Genetika 5: 8-15) despite the fact that the virus is only endemic to the Western Hemisphere.
The shortcomings of the only available EEE vaccine indicate a need for the development of a new formulation. A live attenuated vaccine could offer significant advantages for both human and veterinary use over the inactivated PE-6 preparation. These benefits include administration being limited to a single dose and more efficient production of humoral immunity. A live, attenuated vaccine could also be produced from less starting material than is possible with existing inactivated products. Last, it may provide immunity to EEE that can last for several years or even for life and protect against all strains of EEE.
Deletion of the furin protease cleavage site from VEE and WEE viruses has resulted in the successful production of live-attenuated vaccine candidates. Each of these approaches has been previously patented by others (U.S. Pat. Nos. 6,261,570 and 5,505,947). Research on EEE virus has lagged significantly behind VEE and WEE despite it being the most dangerous of these viruses. To date, there is no known viable EEE vaccine that contains a full or partial deletion of the furin cleavage site.
Therefore, it is an object of the present invention to produce a live attenuated EEE vaccine composed of a furin-cleavage deletion mutant. This vaccine will be derived from a molecular clone of EEE constructed from cDNAs encompassing the entire genome of EEE. This construct will contain a full or partial deletion of the furin cleavage site and/or other deletions within the EEE genome.
The classical method of deriving live-attenuated vaccines involves blind passage of virus in cell cultures. This approach was used successfully to produce alphavirus vaccines for VEE (TC83) and chikungunya (Vaccines, 4th Ed., Chapter 35 Miscellaneous limited-use vaccines, [2004] 991-992). A disadvantage of this approach is that it often results in heterogonous products. We have utilized this classical method in the development of several EEE vaccine candidates. To address any ambiguities attenuated viruses were always sequenced and we subsequently produced defined, molecular clones that behaved comparably to the parental virus.
The alphavirus genome is a single-stranded, positive sense RNA approximately 11,700 nucleotides in length. The 5′ two-thirds of the genome consist of a non-coding region of approximately 46 nucleotides followed by a single open reading frame of approximately 7,500 nucleotides which encodes the viral replicase/transcriptase. The 3′ one-third of the genome encodes the viral structural proteins in the order Capsid-E3-E2-6K-E1, each of which are derived by proteolytic cleavage of the product of a single open reading frame of approximately 3,700 nucleotides. The sequences encoding the structural proteins are transcribed as a 26S mRNA from an internal promoter on the negative sense complement of the viral genome. The nucleocapsid (C) protein possesses autoproteotytic activity which cleaves the C protein from the precursor protein soon after the ribosome transits the junction between the C and E3 protein coding sequence. Subsequently, the envelope glycoproteins E2 and E1 are derived by proteolytic cleavage in association with intracellular membranes and form heterodimers. E2 initially appears in the infected cell as the precursor protein PE2, which consists of E3 and E2. After extensive glycosylation and transit through the endoplasmic reticulum and the golgi apparatus, E3 is cleaved from E2 by the furin protease at a cleavage site having a consensus sequence of RXK/RR, with X being one of many amino acids present in the different viruses and cleavage occurring after the last arginine (R) residue. Subsequently, the E2/E1 complex is transported to the cell surface where it is incorporated into virus budding from the plasma membrane (Strauss and Strauss [1994] Microbiological Rev. 58: 491-562). All documents cited herein supra and infra are hereby incorporated in their entirety by reference thereto. A diagram of the EEE virus genome is shown in FIGS. 3 and 4.
Because the genome of alphaviruses are positive-stranded RNA, and infectious upon transfection of cells in culture, an “infectious clone” approach to vaccine development is particularly suitable. In this approach, a full-length cDNA clone of the viral genome is constructed downstream from a RNA polymerase promoter, such that RNA which is equivalent to the viral genome can be transcribed from the DNA clone in vitro. This permits one to conduct site-directed mutagenesis to insert specific mutations into the DNA clone, which are then reflected in the virus which is recovered by transfection of the RNA.
Previous work with infectious clones of other alphaviruses has demonstrated that disruption of the furin cleavage site results in a virus which incorporates Precursor E2 Protein (PE2) into the mature virus. Davis et al. (1995 supra) found that disruption of the furin cleavage site in an infectious clone of VEE is a lethal mutation. Transfection of BHK cells with RNA transcribed from this mutant clone resulted in the release of non-infectious particles. However, a low level of infectious virus was produced which contained secondary suppressor mutations such that PE2-bearing virus was fully replication competent. It was subsequently shown to be avirulent but capable of eliciting immunity to lethal virus challenge in a variety of animal species.
The genetic basis for attenuation of the VEE TC-83 vaccine and certain laboratory strains of VEE virus (e.g. V3526) have been studied extensively and has led to the development of improved live, attenuated vaccine candidates (Davis et al. 1995, surpa; Pratt et al., [2003] Vaccine 21:3854-3862). The approach used in this application is similar to that used for VEE, however, following the VEE example did not result in an adequate vaccine for EEE. Changes in the procedure used for VEE were required in order to produce the attenuated live EEE virus of the present invention. None of which could have been predicted from previous alphavirus research.
Based upon a comparison of the structural protein gene sequences of EEE and other alphaviruses, the probable furin cleavage site of EEE strain FL91-4697 virus is RRTRR (SEQ ID NO: 2). The presence of the extra arginine when compared to the furin consensus sequence (RX(R/K)R) indicates that cleavage at this site might be more complex than that observed for VEE virus. It was necessary, therefore, to prepare a deletion mutant in the E3-E2 cleavage site of the full-length clone which lacked five amino acids to produce an attenuated virus. The residual arginine in the full-length clone was of concern due to the possibility that other mutations might arise due to the presence of the extra arginine resulting in cleavage by cellular proteases at that site and producing an apparently wild type (wt) virus with respect to cleavage of PE2.
Transfection of cultured cells with RNA transcribed from an infectious clone of EEE lacking either a partial or full deletion of the furin cleavage site yielded viruses which contained the PE2 of EEE in the mature virus but which were not replication competent. During intracellular replication of the RNA, mutations arise at low frequency, resulting in a small number of replication competent viruses. Sequence analysis of these viruses has shown that the lethal effect of the deletion mutations was alleviated by the appearance of second site mutations in the E3, E2 and/or E1 glycoproteins. These viruses are attenuated in mice when administered by subcutaneous or intranasal inoculation. The inoculated mice produced a high titer of serum neutralizing antibody specific to EEE and were protected against a lethal, aerosol challenge of parental virulent EEE virus (>1000×LD50). Similar results were observed in young chickens when mutant EEE viruses were administered by subcutaneous inoculation. The birds produced neutralizing antibody and were protected against a subcutaneous (s.c.) challenge of the parental FL91 EEE virus as well as the South American PE-0155 strain of EEE.
Therefore, in one aspect of the invention, the invention pertains to the isolation of a cDNA sequence coding for wild type infectious eastern equine encephalitis (EEE) virus RNA transcript. DNA representing the entire FL91-4679 genome, not previously available, was prepared by polymerase chain reaction using a series of primer pairs based upon the genome sequence of the PE-6 stain of EEE. In order to determine the correct sequence at the 5′ and 3′ ends of the genome, a protocol called rapid amplification of cDNA ends (RACE) was used. The full length clone is useful in the production of virulent EEE virus and as a platform to introduce and test attenuating mutations. The production of virulent virus is essential for a formal measure of the degree of attenuation achieved with candidate attenuating mutations and a formal determination of the rate at which reversion to virulence might occur.
Portions of the EEE cDNA sequences described above are also useful as probes to diagnose the presence of virus in samples, and to define naturally occurring variants of the virus. These cDNAs also make available polypeptide sequences of EEE antigens encoded within the EEE genome, and permit the production of polypeptides which are useful as standards or reagents in diagnostic tests and/or as components of vaccines. Antibodies, both polyclonal and monoclonal, directed against EEE epitopes contained within this polypeptide sequence would also be useful for diagnostic tests, as therapeutic agents, and for screening of antiviral agents.
Accordingly, with respect to polynucleotides, some aspects of the invention are: a purified EEE polynucleotide, a recombinant EEE polynucleotide to include chimeric viruses; a recombinant polynucleotide comprising a sequence derived from an EEE genome or from EEE cDNA to include a chimeric virus; a recombinant polynucleotide encoding an epitope of EEE, a recombinant vector containing any of the above recombinant polynucleotides, and a host cell transfected with any of these vectors.
Another aspect of the invention is a single-stranded DNA sequence comprising a cDNA clone coding for an infectious EEE, the cDNA clone including at least one attenuating mutation therein, the RNA produced from transcription of the cDNA and the virus particles produced from the RNA in a host cell for use as a vaccine.
In another aspect of the invention there is provided a full length EEE cDNA clone containing a defined deletion mutation useful for attenuating the virus for the identification of suppressor mutations in the virus. The attenuated viruses with the cleavage deletion and suppressor mutations are useful as a means to generate an attenuated, live EEE virus vaccine for veterinary and human use.
In a further aspect of the invention is provided a chimeric virus containing nonstructural and/or structural protein gene sequences derived from EEE and protein gene sequences from any alphaviruses including but no limited to Aura, Barmah Forest, Bebaru, Cabassou, Chikungunya, Everglades, Fort Morgan, Getah, Highlands J, Kyzylagach, Mayaro, Middelburg, Mucambo, Ndumu, O'nyong-nyong, Pixuna, Ross River, Sagiyama, Semliki Forest, SAAR87, Sindbis, Tonate, Una, Venezuelan equine encephalitis, Western equine encephalitis and Whataroa, which could be used as a means for attenuating virulent alphaviruses. Depending on the non-EEE sequences substituted for EEE, another aspect of the invention includes a means to express antigens of other alphaviruses as chimeric alphaviruses as potential vaccines for human and veterinary use.
In a further aspect of the invention, there is provided a vaccine protective against EEE, the vaccine comprising live attenuated EEE virus in an amount effective to elicit protective antibodies in an animal to EEE and a pharmaceutically acceptable diluent, carrier, or excipient.
In yet another aspect of the invention there is provided an inactivated vaccine produced from the live attenuated virus described above. The attenuated virus of the present invention whether whole virus or chimeric virus, can be used in producing inactivated virus vaccines. By using an attenuated EEE virus strain, there is a much greater margin of safety in the event that the product is incompletely inactivated. Starting with an attenuated strain is also much safer during the manufacturing phase, and allows production under lower biocontainment levels.
The live-attenuated EEE vaccine candidates disclosed hereafter are primarily predicated upon deletion of the furin-cleavage site. The present invention has evolved through a 3-tier approach to produce the candidates disclosed and claimed herein.
These and other objects will become apparent upon further reading of this disclosure. In this application are described live attenuated vaccines for EEE which may provide higher level immunity in humans.